FULL PAPER Pharmacology
Synergistic growth inhibitory effect of deracoxib with doxorubicin against a canine mammary tumor cell line, CMT-U27
Tülay BAKIREL1)*, Fulya Üstün ALKAN1), Oya ÜSTÜNER1), Suzan ÇINAR2), Funda YILDIRIM3), Gaye ERTEN2) and Utku BAKIREL4)
1)Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul University, Istanbul, 34320, Turkey 2)Department of Immunology, Institute of Experimental Medicine, Istanbul University, Istanbul, 34093, Turkey 3)Department of Pathology, Faculty of Veterinary Medicine, Istanbul University, Istanbul, 34320, Turkey 4)Department of Internal Medicine, Faculty of Veterinary Medicine, Istanbul University, Istanbul, 34320, Turkey
(Received 2 July 2015/Accepted 13 December 2015/Published online in J-STAGE 29 January 2016)
ABSTRACT. Cyclooxygenase (COX) inhibitors have been shown to exert anti-angiogenic and anti-tumor activities on many types of ma- lignant tumors. These anticancer properties make it worthwhile to examine the possible benefit of combining COX inhibitors with other anti-cancer agents. In the present study, we evaluated the potential of deracoxib (DER) in potentiating antitumor activity of doxorubicin (DOX) in canine mammary carcinoma cells (CMT-U27). DER (50–250 µM) enhanced the antiproliferative activity of DOX by reducing the IC50 (approximately 3- to 3.5 fold). Interaction analysis of the data showed that combinations of DOX at 0.9 µM with DER (100–250 µM) produced synergism in the CMT-U27 cell line, with a ratio index ranging from 1.98 to 2.33. In additional studies identifying the mechanism of observed synergistic effect, we found that DER strongly potentiated DOX-caused G0/G1 arrest in cell cycle progression. Also, DER (100–250 µM) augmented apoptosis induction with approximately 1.35- and 1.37- fold increases in apoptotic response caused by DOX in the cells. DER enhanced the antiproliferative effect of DOX in conjunction with induction of apoptosis by modulation of Bcl-2 expression and changes in the cell cycle of the CMT-U27 cell line. Although the exact molecular mechanism of the alterations in the cell cycle and apoptosis observed with DER and DOX combinations require further investigations, the results suggest that the synergistic effect of DOX and DER combinations in CMT therapy may be achieved at relatively lower doses of DOX with lesser side effects. Therefore, combining DER with DOX may prove beneficial in the clinical treatment of canine mammary cancer. KEY WORDS: apoptosis, canine mammary tumor, cell cycle, deracoxib, doxorubicin doi: 10.1292/jvms.15-0387; J. Vet. Med. Sci. 78(4): 657–668, 2016
Canine mammary tumors (CMTs) are the most common (DOX), either alone or in combination with other drugs, is a neoplasms occurring in female dogs, with a prevalence of widely used anti-cancer agent for the therapy of these tumors 0.2% [23, 27]. Histologically, approximately 50–71% of in veterinary practice [33, 66]. It is known that failure of che- these neoplasms are considered malignant, and distant me- motherapy with this agent as a result of development of re- tastases are common causes of death in cancer patients [67]. sistance and dose-limiting toxicity is a major problem in the Despite their importance and high incidence, their molecular clinical management of the neoplasms [39, 62]. Therefore, mechanisms have not been completely defined, and effec- alternative strategies that increase the therapeutic efficacy tive therapeutic options are scarce [26, 43]. The preferred and minimize the systemic toxicity of the chemotherapeutic therapy for CMT is surgical intervention with the intention agent against mammary tumors in dogs are needed. of tumor extirpation, but it alone yields unsatisfactory results Numerous experimental, epidemiological and clini- in dogs with malignant mammary tumor, because scattered cal studies indicate that nonsteroidal anti-inflammatory tumor cells and metastases cannot be eliminated [19, 59, 64]. drugs (NSAIDs), particularly the selective cyclooxygenase At present, the use of anticancer drugs to combat the mi- (COX)-2 inhibitors, have chemotherapeutic and chemo- crometastatic disease is a reasonable consideration, but there prophylactic potentials in several different types of cancer, are no agents currently approved for the treatment of malig- including breast carcinoma. Antitumorigenic and chemo- nant mammary tumors in dogs. Many of the chemotherapy therapeutic effects of these inhibitors have been attributed protocols used in veterinary medicine have been co-opted to both COX-dependent and COX-independent mechanisms from protocols used to treat human patients [41, 60]. Among relating to induction of cell apoptosis and inhibition of an- the several types of chemotherapeutic drugs, doxorubicin giogenesis and cell invasion/migration [4, 6, 20, 42, 49, 57]. Also, numerous investigators have explored the therapeutic benefit of combining selective COX-2 inhibitors with che- *Correspondence to: Bakirel, T., Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Istanbul Univer- motherapeutics and have shown that the COX-2 inhibitors sity, 34320, Istanbul, Turkey. e-mail: [email protected] are able to enhance the effects of certain cytostatic agents, ©2016 The Japanese Society of Veterinary Science such as doxorubicin, irinotecan and 5-fluorouracil in vari- This is an open-access article distributed under the terms of the Creative ous human carcinoma cell lines and in a xenograft animal Commons Attribution Non-Commercial No Derivatives (by-nc-nd) model [6, 22, 61, 69]. Currently, a number of phase I-III License
COX-2 inhibitors as single agents or in combination with a humidified atmosphere at 37°C under 5% CO2 and 95% air chemotherapy in cancer therapy [16, 50]. In veterinary to allow cell adhesion. After 24 hr incubation, the medium medicine, data from experimental and clinic studies exist on was removed, and cells were treated with various concentra- the efficacy of selective COX-2 inhibitors (firocoxib, dera- tions of DOX (0.1, 1, 10, 50 and 100 µM) and DER (50, 100 coxib) against CMT [2, 46], but to our knowledge there is no and 250 µM) for 72 hr. The concentrations for DOX were published report demonstrating synergistic cytotoxic effects chosen on the basis of previous reports about the effects of on canine mammary carcinoma cells elicited by anticancer this drug on the in vitro viability of canine mammary tumor agents in combination with these drugs. If synergistic effects cells [39]. DER concentrations were selected according to could be achieved by combining selective COX-2 inhibitors our previous study [2]. Cell viability, based on mitochondrial with anticancer agents for CMTs, as well as in some types dehydrogenase activity, was determined by colorimetric of human cancer [6, 17, 25, 47], they could reduce the doses assay using a MTT Cell Proliferation Kit (Roche Applied of anticancer drugs needed and thus occurrence of compli- Science, Mannheim, Germany) in accordance with the in- cations. Therefore, we decided to investigate whether the struction manual. The optical density of each well at 550 selective COX-2 inhibitor deracoxib (DER), a drug licensed nm against a reference wavelength of 650 nm was measured for veterinary use, could potentiate the inhibitory action of using a microplate reader (ELx800, Biotek Instruments, DOX, the standard chemotherapeutic drug, in a canine mam- Winooski, VT, U.S.A.). Cell viability was calculated as fol- mary tumor cell line (CMT-U27). lows: Viability (%)=(Absorbance of the treated wells)/(Ab- sorbance of the control wells) ×100. Each concentration was MATERIALS AND METHODS tested in three different experiments and run in triplicate. The dose-response curves were plotted for each drug, and Cell line and chemicals: To investigate the potential ef- the concentration of drug required for 50% inhibition of cell fects of the selective COX-2 inhibitor DER and cytotoxic viability (IC50) was determined graphically. Subsequently, anthracycline DOX against canine mammary carcinoma we tested 0.9 µM (IC50) and 0.09 µM (1/10 IC50) of DOX cells, the CMT-U27 cell line was chosen, as it has high pro- with 50, 100 and 250 µM of DER in combination to learn liferative and anti-apoptotic potential [29]. The canine mam- whether DER could enhance the antiproliferative effects of mary carcinoma cell line (CMT-U27) was kindly supplied by DOX in CMT-U27 cells. For this purpose, the cells were Prof. Eva Héllmen, Uppsala University, Sweden. This cell treated with DOX and DER for 72 hr, and the antiprolifera- line was derived from a primary tumor (infiltrating ductal tive effects of the combined agents were evaluated according carcinoma), and when inoculated in the fat mammary pad of to the MTT assay. female nude mice, it metastasized to the lymph nodes, lungs, Drug interaction analysis: The nature of the interaction liver and heart [18]. DER was a generous gift from Novar- between DOX (0.9 µM and 0.09 µM) and DER (50, 100 and tis Pharmaceuticals Inc. (Basel, Switzerland). Polyclonal 250 µM) was determined by calculating the ratio index (RI), anti-Bcl-2 (sc-492), antibody, a secondary antibody kit and which was initially described by Kern et al. [24] and later 3,3′-diaminobenzidine (DAB) (sc-2018) were purchased modified by Romanelliet al. [53]. The RI is calculated as the from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Un- ratio of expected cell survival (Sexp, defined as the product less otherwise indicated, all reagents were purchased from of the viability observed with drug A alone and the survival Sigma-Aldrich (St. Louis, MO, U.S.A.). observed with drug B alone) to the observed cell survival Cell culture: Cells were cultured in Dulbecco’s modified (Sobs) for the combination of A and B (RI=Sexp/Sobs). Type eagle’s medium (DMEM-F12) supplemented with 10% fetal of interaction was defined as follows: RI≥1.5, synergistic; bovine serum, 100 IUml−1 penicillin G, 100 µgml−1 strep- RI<1.5 to >0.5, additive; and RI≤0.5, antagonistic [31]. This tomycin and 2.5 µgml−1 amphotericin B in an atmosphere method was selected, because treatment with DER had little of 37°C in 5% CO2, and the cells were harvested at ap- effect on cell viability, which meant that other methods, such proximately 80–90% confluence using 0.25% trypsin-EDTA as the median effect principle and isobologram methods, solution. DOX and DER were dissolved in DMEM-F12 and were not suitable. sterile dimethyl sulfoxide (DMSO), respectively, and further Apoptosis assay: To determine the mechanism of inter- serial dilutions for both drugs were made with DMEM-F12. action between DER and DOX, an apoptosis assay was The final DMSO concentration did not exceed 0.1% (and performed. Flow cytometric analyses of phosphatidylserine had no effect on cell growth) in any experimental group, and exposure were quantitatively performed using an Annexin this condition was used as a control in each experiment (all V-fluorescein isothiocyanate (FITC) Apoptosis Detection groups comprised 0.1% DMSO). All of the stock solutions Kit (BD Biosciences, San Jose, CA, U.S.A.). The method were kept at −20°C. is based on the binding of Annexin V to phosphatidylserine Cell viability assay: To determine DOX and DER con- that is translocated from the inner membrane leaflet to the centrations that would be used in combination studies, an outer layer in cells undergoing apoptosis [68]. The cells were MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium cultured at a density of 1 × 105/ml in 24-well flat bottom bromide) tetrazolium reduction assay was performed on microtiter plates (Jet Biofil, Seoul, Korea) and cultivated in a CMT-U27 cells. For this purpose, cells were seeded at 1 × medium as described above. After 24 hr, the medium was re- 104/well in a final volume of 100 µl medium in 96-well flat- placed with fresh medium containing DOX (0.9 µM) with or bottomed tissue culture plates, in triplicate, and incubated in without DER (50–250 µM). The cells were trypsinized 72 hr INTERACTION OF DERACOXIB WITH DOXORUBICIN 659 after treatment, washed twice each with ice-cold phosphate- Afterwards, cells were incubated with polyclonal anti-Bcl-2 buffered saline (PBS) consisting of 0.01 M phosphate buffer, (sc-492) antibody at a dilution of 1:200 at room temperature 0.0027 M potassium chloride and 0.137 M sodium chloride for 90 min. After extensive washing in PBS, the cover slips and then resuspended in 100 µl binding buffer (0.1 M Hepes/ were incubated with a secondary antibody kit (sc-2018) NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2), additive sup- containing a biotinylated secondary antibody and avidin- plemented with 5 µl of FITC-Annexin V and 5 µl of prop- peroxidase for 20 min, at room temperature. Finally, cells idium iodide (PI). The cell suspension was gently vortexed were rinsed with PBS and incubated with DAB complexes and incubated for 15 min at room temperature in the dark. according to the manufacturer’s protocol and then counter- Following incubation, 400 µl of binding buffer was added to stained with Mayer’s hematoxylin and mounted onto glass each tube, and then, the cell suspension was analyzed within slides. The cells were observed with a light microscope 1 hr on a FACScan flow cytometer (BD Biosciences) using (Olympus BX50, Olympus, Tokyo, Japan). Intensity of the standard optics for detecting FL1 (FITC) and FL3 (PI). immunolabeling was assessed by examination of 10 rep- Data were analyzed with the CellQuest WinMDI software resentative high-power fields (400 ×). Positive cells were (BD Biosciences, San Jose, CA, U.S.A.). identified by distinct brown nuclear staining. The number of Cell cycle analysis: The effects of DOX (0.9 and 0.09 µM) immunoreactive cells was assessed semiquantitatively. For with or without DER (50–250 µM) on the CMT-U27 cell staining density, specimens were classified as negative, + cycle were evaluated by flow cytometry using a Coulter (<10% positive cells), ++ (10–50% positive cells) and +++ DNA Prep Reagents Kit (Beckman Coulter, High Wycombe, (>50% positive cells). Results were considered inconclusive Buckinghamshire, U.K.). The cells were cultured at a den- when there were insufficient neoplastic cells available for sity of 1 × 105/ml in 24-well flat bottom microtiter plates analysis [73]. and cultivated and treated as described for an apoptosis Statistical analysis: Samples were assayed at least three assay. After the 72 hr treatment, the floating and adherent times for each determination, and results were expressed as cells were combined for the analyses. Cells were washed the mean ± SE. The statistical differences between the treat- with PBS, and the cell suspensions were resuspended in 100 ments and the control were tested by one-way analysis of µl of PBS. The resuspended cells were stained according variance (ANOVA) followed by the Student’s t-test using the to the manufacturer’s instructions. The DNA content of the GraphPad InStat software (GraphPad Software, San Diego, stained cells was immediately analyzed using a FACScan CA, U.S.A.). A difference in means with a P-value of 0.05 or flow cytometer (BD Biosciences). At least 10,000 cells were less was considered to be statistically significant. counted. The percentages of cells in the G0/G1 phase, S phase and G2/M phase were calculated using the CellQuest RESULTS WinMDI software (BD Biosciences). Prostaglandin E2 (PGE2 assay): In order to elucidate Antiproliferative effects of DER and DOX combinations whether the antiproliferative effect was mediated via the on CMT-U27 cells: The antiproliferative effects of DER and COX pathway, we studied the effect of exogenous addi- DOX combinations on CMT-U27 cells were evaluated by tion of PGE2 (1–5 µg/ml) on the in vitro antiproliferative MTT assay. The antiproliferative effects of DER and DOX activity of DER alone and in combination with DOX against as single agents against CMT-U27 cells were shown in our CMT-U27 cells. The cells were seeded at 1 × 104/well in previous studies [2, 3]. Those studies demonstrated that DER 100 µl of medium in 96-well plates and incubated over- even at concentration as high as 250 µM had weak growth night. Subsequently, DOX (0.9 and 0.09 µM) was added to inhibitory activity in the cells (with growth inhibition of the plate in the presence or absence of PGE2 (1–5 µg/ml). 16.49% compared with the control). By contrast, CMT-U27 Similarly, DER (50–250 µM) in combination with DOX (0.9 cells responded to DOX sensitively, with an obvious dose- and 0.09 µM) was added in the presence or absence of PGE2 dependent antiproliferative effect and IC50 of approximately (1–5 µg/ml) and incubated for 72 hr, and the antiproliferative 0.9 µM. Subsequently, to determine whether DER enhances activity was assessed by MTT assay. the anti-proliferative effect of DOX on CMT-U27 cells, Immunocytochemistry: In order to examine the protein three doses of DER (50, 100 and 250 µM) were used in com- expression of the apoptosis marker Bcl-2 in CMT-U27 cells, bination with DOX (0.9 and 0.09 µM). As shown in Fig. 1, sterilized coverslips were placed on the bottom of a 24-well DER potentiated the inhibitory effect of DOX (0.9 µM) on plate, and the cells were seeded at a density of 1 × 105 cells/ CMT-U27 cells. The influence of DER on the effect of DOX ml and incubated overnight. After incubation, cells were (0.9 µM) appeared to be moderately dose dependent, and the treated with 50–250 µM DER alone or in combination with strongest inhibition, approaching 71%, was observed with 0.9 µM and 0.09 µM DOX for 72 hr. At the end of treat- the combination of DOX at 0.9 µM and DER at 250 µM. ment, the cells were rinsed with PBS and fixed with cold Also, the COX inhibitor at the concentrations used enhanced methanol for 10 min. After fixation, the cells were washed the antiproliferative activity of DOX in CMT-U27 cells, with PBS (pH 7.4, 0.1 M) for 5 min, and the endogenous which decreased (2.89- to 3.71-fold) the IC50 value from peroxidase activity was inactivated by incubation with 0.3% 0.876 µM (DOX at 0.9 µM) to 0.303 µM, 0.245 µM and H2O2 in methanol for 10 min. Then, the cells were washed 0.236 µM (0.9 µM in combination with 50, 100 and 250 µM three times with PBS and incubated for 10 min with a pro- DER), respectively. tein blocking agent to block nonspecific immunolabeling. The nature of the interaction between DOX and DER was 660 T. BAKIREL ET AL.
Fig. 1. Antiproliferative effects of doxorubicin and deracoxib combinations in the CMT-U27 cell line. Cells were treated with the indicated doses of doxorubicin and deracoxib, and cell viability was assayed 72 hr after treatment. Data are expressed as mean percentage of cell viabilities ± standard error (SE). **P<0.01 compared with the doxorubicin-treated group. DOX, doxorubicin; DER, deracoxib. analyzed using the RI, which quantitatively measures the Table 1. Type of interaction between doxorubicin interaction of two drugs. The combinations of DOX at 0.9 and deracoxib in CMT-U27 cells µM and DER at 50–250 µM exhibited synergistic activity Combination of DOX with DER RI value against CMT-U27 cells (Table 1). The synergistic effect was DOX (0.9 µM) most prominent when 0.9 µM DOX was combined with 250 DOX+ DER (50 µM) 2.24 (synergistic) µM DER (RI=2.33). On the other hand, the combinations DOX+ DER (100 µM) 1.98 (synergistic) of a low dose (0.09 µM) of DOX with DER (50–250 µM) DOX+ DER (250 µM) 2.33 (synergistic) resulted in additive interaction (RI=1.08–1.15) DOX (0.09 µM) Effects of DER and DOX combinations on apoptosis in DOX+ DER (50 µM) 1.15 (additive) CMT-U27 cells: The apoptotic effects of DER as a single DOX+ DER (100 µM) 1.13 (additive) agent against CMT-U27 cells were shown in our previous DOX+ DER (250 µM) 1.08 (additive) study [2]. To explore the mechanisms of synergistic ef- DOX, doxorubicin; DER, deracoxib; RI, ratio index. fects of DOX and DER combinations, an apoptosis assay Type of interaction: RI≥1.5, synergistic; RI<1.5 to >0.5, was performed using specific concentrations of DOX (0.9 additive; RI≤0.5, antagonistic interaction. µM) and DER (50–250 µM) that cannot lead to toxicity. Treatment with DOX (0.9 µM) alone induced 36.22% apop- tosis in CMT-U27 cells. The combined treatment of DOX 0.9 µM DOX and 250 µM DER. This combination led to a and DER (100 µM and 250 µM) significantly augmented further increase in the G0/G1 phase (from 56.6 to 91.34%) apoptosis induction in CMT-U27 cells (sum of early and with a concomitant decrease in the S phase (from 34.83 to late apoptotic cells; 48.91% and 49.58%, respectively), and 7.16) compared with DOX alone (0.9 µM). approximately 1.35- and 1.37-fold increases, respectively, Effects of supplementation of PGE2 on growth inhibitory in apoptotic response were observed as compared with that activity of DOX enhanced by DER in CMT-U27 cells: The caused by DOX. Treatment with DOX (0.09 µM) alone in- effects of supplementation of PGE2 on the growth inhibitory duced 24.02% apoptosis in CMT-U27 cells. The combined activity of DOX enhanced by DER in CMT-U27 cells may treatment of DOX (at 0.09 µM) and DER (at 50 µM, 100 µM be due to COX-2 inhibition. To examine this, we determined and 250 µM) induced 27.77%, 35% and 37.69% apoptosis, whether the COX-2 end product PGE2 could reverse the respectively, in CMT-U27 cells in terms of the sum of early observed effects. Therefore, exogenous PGE2 (1–5 µM) was and late apoptotic cells (Fig. 2A and 2B). added to the medium in order to take into account the fact Effects of DOX and DER combinations on the cell cycle that some PGE2 may degrade or be internalized into cells. distribution of CMT-U27 cells: To confirm the mechanism of As shown in Fig. 3A and 3B, cell viability was not changed such synergistic effects of DOX and DER combinations, we significantly with the addition of PGE2. PGE2 at all tested also examined the effects of their combinations on cell cycle concentrations failed to reverse the enhancing effect of DER parameters under the same experimental conditions. As on the growth inhibitory activity of DOX. shown in Table 2, the percentage of cells in the G0/G1 phase Effects of DOX and DER alone or in combination on Bcl- and S phase of the cell cycle increased with DOX (0.9 µM) 2 expression in CMT U27 cells: To determine the apoptotic treatment as a single agent, while the proportion of cells in pathway activated by DER, we examined the apoptosis-re- the G2/M phase decreased compared with the control. The lated target Bcl-2 in CMT-U27 cells. Immunocytochemical combined treatments of DOX and DER altered the cell cycle staining revealed that increasing the concentrations of DER profile in CMT-U27 cells. The most remarkable change in inhibited expression of the anti-apoptotic protein Bcl-2 cell cycle progression was seen with the combination of (Fig. 4). These effects were more pronounced in the com- INTERACTION OF DERACOXIB WITH DOXORUBICIN 661
Fig. 2. Effects of doxorubicin and deracoxib combination treatment on apoptosis of CMT-U27 cells. (A) The number of viable, early apoptotic, late apoptotic and necrotic cells due to treatment with doxorubicin and deracoxib combinations for 72 hr in the CMT-U27 cell line. The experiment was conducted in three replicates. Data are expressed as the mean ± standard error (SE). *P<0.05 compared with the 0.9 µM doxorubicin-treated group. DOX, doxorubicin; DER, deracoxib. (B) Representative cytograms of the CMT-U27 cell line double stained with Annexin V-FITC and propidium iodide (PI). The numbers written on histograms represent the sum of early and late apoptotic cells (%). 662 T. BAKIREL ET AL.
Table 2. Effects of doxorubicin and deracoxib combination treatment for 72 hr on cell cycle kinetics of CMT-U27 cells
Drugs Concentration G0/G1 (%) S (%) G2/M (%) Control - 54.51 ± 2.68 29.05 ± 1.68 16.44 ± 1.24 DOX 0.9 µM 56.6 ± 4.24 34.83 ± 2.53 8.51 ± 1.14 DOX + DER 0.9 µM + 50 µM 84.49 ± 3.60 15.30 ± 2.08 0.21 ± 0.04a) DOX + DER 0.9 µM + 100 µM 84.96 ± 1.45 14.83 ± 2.65 0.21 ± 0.03a) DOX + DER 0.9 µM + 250 µM 91.34 ± 3.64 7.16 ± 1.32a) 1.49 ± 0.18a) DOX 0.09 µM 86.93 ± 4.99 12.92 ± 1.72 0.14 ± 0.04 DOX + DER 0.09 µM + 50 µM 89.58 ± 5.46 10.30 ± 1.06 0.12 ± 0.03 DOX + DER 0.09 µM + 100 µM 89.13 ± 4.49 10.84 ± 1.50 0.20 ± 0.04 DOX + DER 0.09 µM + 250 µM 89.79 ± 2.85 10.1 ± 1.46 0.10 ± 0.02 Each value represents the mean ± SE of three experiments. a) P<0.05 compared with the 0.9 µM doxorubicin-treated group. DOX, doxorubicin; DER, deracoxib.
DISCUSSION
Despite many advances in the field of cancer therapeutics, canine malignant mammary tumors continue to be a leading cause of death in dogs, which is in part due to the failure of chemotherapy. Clearly, new therapeutic modalities are need- ed to both improve the outcome of dogs suffering from the tumors and to reduce the long-term toxicities associated with the current standard of treatment. Drug development strate- gies include the evaluation of new drug combinations that may have improved efficacy compared with single agents. By treating a tumor with a combination of agents that employ different mechanisms of action and have different spectra of normal tissue toxicity, the overall response can be enhanced without an increase in toxicity [35, 37]. Besides enhanced cytotoxicity, combinations of chemotherapeutic agents may minimize or delay the induction of drug resistance [10, 21]. Furthermore, drug combinations that use lower doses of individual agents may improve selectivity and reduce the severity of undesired side effects of chemotherapy [45]. Selective COX-2 inhibitors exert antitumor effects on tu- mor cells directly via inhibition of cell proliferation, induc- tion of apoptosis and reduction of cell motility and adhesion and also have anti-angiogenic activity by suppressing tumor angiogenesis [36, 56, 69, 72]. These anticancer proper- ties make it worthwhile to examine the possible benefit of combining selective COX-2 inhibitors with conventional anticancer therapies, such as chemotherapy. Related studies have shown that selective COX-2 inhibitors in particular strengthen the effectiveness of chemotherapy treatment in Fig. 3. Effects of supplementation of PGE2 on enhancement of various types of human tumors, including breast tumors [5, growth inhibitory activity of doxorubicin caused by deracoxib 9, 56, 69]. Similarly, during the last decade, various preclini- in CMT-U27 cells. Cells were treated with or without deracoxib cal and clinical trials in the field of veterinary oncology have (50-250 µM) in the absence or presence of PGE2 (1 or 5 µM) and 0.9 µM doxorubicin (A) or 0.09 µM doxorubicin (B) alone or in been conducted to study the use of COX-2 selective inhibi- combination for 72 hr before determination of cell viability by tors in combination with other agents in early and advanced MTT assay. DOX, doxorubicin; DER, deracoxib. cancers and have been shown to improve treatment outcome in dogs with transitional cell carcinoma and osteosarcoma [34, 70]. However, to our knowledge, there is no published binations of DOX (at 0.9 µM) with DER (at 100 µM and report demonstrating the therapeutic efficiency of selective 250 µM) (Table 3). However, the combinations of DOX at COX-2 inhibitors with conventional anti-cancer agents in 0.09 µM with DER (at 100 µM and 250 µM) decreased the canine mammary tumor. Based on the encouraging results expression of Bcl-2 slightly. from COX-2 inhibitors as chemotherapeutic and chemopre- INTERACTION OF DERACOXIB WITH DOXORUBICIN 663
Fig. 4. Immunocytochemical expression of Bcl-2 in CMT U27 cells. A) Deracoxib 50 µM, moder- ate immunopositivity; B) Deracoxib 250 µM, slight immunopositivity; C) Doxorubicin 0.09 µM + deracoxib 50 µM, strong immunopositivity; D) Doxorubicin 0.9 µM + deracoxib 250 µM slight immunopositivity; E) Control cells, very strong immunopositivity; F) Negative control cells, no reac- tion. Bar=20 µm. ventive agents in the treatment of several types of human [34, 54]. Similarly, in our previous investigation, we proved and canine malignancy including mammary tumor [2, 14], that DER had a clear antiproliferative and apoptotic effect on we evaluated the potential of a selective COX-2 inhibitor canine mammary carcinoma cells in vitro [2]. These effects (DER) in potentiating the antitumor activity of a conven- have only been observed at high concentrations (250–1,000 tional cytotoxic agent (DOX) in vitro in canine mammary µM) of DER as well as seen in other NSAIDs (e.g., indo- carcinoma cells (CMT-U27). For this purpose, we preferred methacin, meloxicam), which are 10- to 1,000-fold those DER, a highly selective canine COX-2 inhibitor accepted as required to inhibit PG synthesis or COX enzymes [38, safe and well-tolerated in dogs [52], and DOX, a cytotoxic 51, 63]. The clinical importance of the direct cytotoxicity anthracycline antibiotic commonly used in veterinary clini- observed in vitro is presently unknown, as it is not known cal treatments for various cancers [62]. what plasma concentrations of DER would be necessary to DER is widely used in veterinary medicine for the con- achieve effective concentrations (≤250 µM) intratumorally trol of pain and inflammation associated with osteoarthritis or what duration of drug exposure is necessary to induce and orthopedic surgery in dogs [8]. Recently, it has been the observed cytotoxicity. However, it is known that plasma reported that this drug might be a useful alternative for the concentrations of DER can reach as much as 20 µM when prevention and/or treatment of some cancer types in dogs DER is administrated at a dose of 4 mg/kg daily (i.e. the dos- 664 T. BAKIREL ET AL.
Table 3. Effects of deracoxib or doxorubicin alone was within the range of clinically relevant concentrations, as or in combination on Bcl-2 expression in CMT- dogs that were treated with 1 mg/kg DOX achieved plasma U27 cells concentrations of 0.7 µg/ml (1.2 µM) 5 min after intrave- Drugs Concentration Bcl-2 nous administration [70]. The concentrations of DOX for a Control - +++ desirable pharmacological effect in canine mammary tumor DOX 0.9 µM ++ cell lines in vitro were reported to be 280 nM and 840 nM DER 50 µM ++ [58, 70]. The IC50 value for CMT-U27 cells is higher than DER 100 µM + reported for the same compound in different CMT cells. DER 250 µM + We suggest that DOX is less potent in CMT-U27 cells than DOX + DER 0.9 µM + 50 µM ++ other mammary tumor cell types. CMT-U27 cells have a DOX + DER 0.9 µM + 100 µM + high growth rate and anti-apoptotic potential associated with DOX + DER 0.9 µM + 250 µM + enhanced expression of genes involved in the Ca2+ signaling DOX 0.09 µM +++ pathway and growth hormone cellular pathway [68]. The DOX + DER 0.09 µM + 50 µM +++ high anti-apoptotic potential of these cells has been shown to DOX + DER 0.09 µM + 100 µM ++ be related to elevated expression ABR, which interacts with DOX + DER 0.09 µM + 250 µM ++ the tumor protein P53 and TMD1 genes, which are involved DOX, doxorubicin; DER, deracoxib. Results are in drug resistance in tumor cells [30]. Prior studies have also categorized as negative, + (<10% positive cells), + + demonstrated that CMT-U27 cells express high levels of (10–50% positive cells) and +++ (>50% positive cells). anti-apoptotic Bcl-2 protein, which is linked to resistance to several therapeutic modalities [30, 68]. These data confirm our finding that highly metastatic CMT-U27 cells have low age used for postoperative orthopedic pain) [54]. It is also sensitivity to DOX. Therefore, we concluded that CMT-U27 accepted that use of the drug at higher than approved dos- cells could be an in vitro model for canine mammary cancer ages, especially long-term use, can lead to an increased risk refractory to DOX chemotherapy. of toxicity [48]. On the other hand, the concentration in the In this study, we tested whether a COX-2 inhibitor drug tumor may differ from that in plasma. It has been reported could restore the response of a chemotherapeutic agent in that the concentrations of NSAIDs, extensively bound to canine mammary cancer. Our results demonstrated that DER proteins, in the acidic environment of inflammation (such as enhanced the cytotoxic action of DOX at 0.9 µM in CMT- in a tumor) relating to high protein content might be higher U27 cells in a dose dependent manner, which decreased the than in plasma [28, 70]. Similarly, tumor cells are able to IC50 value from 0.876 µM (DOX at 0.9 µM) to 0.303 µM, produce angiogenic proteins like the vascular endothelial 0.245 µM and 0.236 µM (0.9 µM in combination with 50, growth factor (VEGF), which could lead to a preferential 100 and 250 µM DER), respectively. These results suggested accumulation of NSAIDs, so it is possible that the concentra- that DER sensitized CMT-U27 cells to the action of DOX. tions in the tumor cells are higher than in plasma [70]. Im- The addition of DER to 0.9 µM DOX, even at concentra- munohistochemical studies have demonstrated that VEGF is tions that did not affect cell viability, achieved at least the highly expressed in CMT cells [27]. It has been reported that same level of cytotoxicity as an approximately 3- to 3.5- VEGF and a beta-galactoside-binding protein (galectin-3, an fold higher dose of DOX alone in the cell line. In contrast, important mediator of VEGF) are highly expressed in CMT- DER exerted no effect on the action of DOX at 0.09 µM U27 cells [7]. Based on this information, we used the lowest in the cell line. These results suggest that the enhancement possible concentrations (50 µM, 100 µM and 250 µM) of of cytotoxicity in combination treatment of DOX with DER DER, which are likely the most practical for clinical use, in depends on the DOX chemotherapy dose and that the addi- our combination studies. tion of DER (50–250 µM) to DOX (0.9 µM) therapy may DOX is a cytotoxic anthracycline antibiotic that is com- be beneficial to reduce the chemotherapy dose necessary to monly used in both veterinary and human cancer chemo- achieve a cytotoxic response in canine mammary cancer. To therapy protocols [40, 62]. This medication has antitumor determine the nature of the interaction between DOX and activity against a wide variety of carcinomas including DER, we used the method of Kern et al. as modified by Ro- mammary tumors, however, its utility is limited due to acute manelli et al. [24, 53]. This method is accepted as the only and chronic toxicities, such as myelosuppression, immuno- correct method to evaluate the type of interaction between suppression and dose-cumulative cardiotoxicity [11, 66]. two drugs when one or both has a low cytotoxic effect or Therefore, it is important to use this drug in in vitro thera- no dose-response curve [31]. Interaction analysis of the data peutic tests with the purpose of searching for a new method- showed that the combinations of DOX at 0.9 µM with DER ology for combination protocols with nontoxic drugs, as it produced synergism in the CMT-U27 cell line, with a ratio could enable the dose of DOX to be lowered due to its good index ranging from 1.98 to 2.33. DOX at a low concentra- antitumor activity, which is mainly observed in the meta- tion (0.09 µM) with DER exhibited interaction in an additive static mammary tumor. Our previous study indicated that a manner. Our results are consistent with the work of Van Wi- broad concentration range (0.1–100 µM) of DOX inhibited jngaarden et al. [69], who demonstrated that celecoxib en- canine mammary carcinoma cell proliferation in vitro [3]. hanced DOX-induced cytotoxicity in MDA-MB231 breast The IC50 of 876 nM determined for DOX in CMT-U27 cells cancer cells. These effects are likely to be mediated via a INTERACTION OF DERACOXIB WITH DOXORUBICIN 665
COX-independent mechanism. The induction of COX-2 and which is known to promote cell survival and has been corre- its associated production of PGE2 from arachidonic acid are lated with the development of DOX resistance [1], in CMT- thought to play a role in the initiation and maintenance of U27 cells. We showed that DER (100 and 250 µM) induced cancer cell survival and growth [17]. The downstream prod- downregulation of Bcl-2 at 0.9 µM DOX more strongly than uct of the COX-2 catalyzed metabolism of arachidonic acid, at 0.09 µM DOX in CMT-U27 cells. We concluded that DER PGE2, has been shown to promote the growth of breast car- may be altered the sensitivity of CMT-U27 cells to DOX- cinoma cells and reverse the growth inhibitory effect of the induced apoptosis by downregulating Bcl-2 expression. selective COX-2 inhibitor celecoxib in breast cancer MCF-7 Other possible functions of DER relating to anti-apoptotic cells [12]. In our study, we found that exogenous addition of genes need to be further investigated. PGE2 (1–5 µg/ml) did not reverse the antiproliferative effect In additional studies identifying the mechanism of ob- of DER in combination with DOX. These results suggest served synergistic and additive effects in treatment with DOX that growth inhibition in CMT-U27 cells by DOX and its and DER, we found that DER potentiated DOX-caused G0/ combination with DER are not directly associated with their G1 arrest in cell cycle progression, while DOX-induced cell ability to suppress PGE2 production. Our results are sup- cycle arrest in the G0/G1 phase, as well as induction of cell ported by the data of Duffy et al. (1998), who showed that death, has been demonstrated in breast cancer MCF-7 cells exogenous PGE2 did not reverse the synergistic cytotoxicity [55]. We showed that a low concentration of DOX (0.09 µM) of DOX with indomethacin against a DLKP cell line [15]. induced higher G0/G1 arrest compared with a high concen- Similarly, PGE2 and the prostaglandin precursor arachidonic tration of DOX (0.9 µM). Similar effects were observed in acid were shown to not reverse the growth inhibitory ef- the combination study. The current results indicated that cell fects of the COX-2-selective inhibitor SC236 with DOX cycle regulation by DOX occurs differentially according to on HKESC-1 and HKESC-2 cells [71]. These observations the concentrations applied. This effect could result from the suggest that COX-independent mechanisms are responsible increased penetration of DOX into CMT-U27 cells caused for the in vitro growth inhibitory effects of these compounds. by DER. On the other hand, the percentages of CMT-U27 The synergistic and additive effects on CMT-U27 cells elic- cells arrested at the G0/G1 phase are inversely proportional to ited by treatment with DOX (0.9 and 0.09 µM, respectively) the DOX concentrations, and this could arise from the den- and DER (especially at the concentrations of 100 and 250 sity of the viable cells observed with both DOX treatments, µM) are associated with a marked increase in apoptosis. Our because high concentrations of DOX lead to necrosis. This results demonstrated that DER (100 and 250 µM) enhanced is the first study to demonstrate that DOX and DOX with the apoptotic activity of DOX at 0.9 and 0.09 µM in the DER induced cell cycle arrest in CMT-U27 cells, and the cells, which increased the apoptotic index from 36.22% to molecular mechanism of accumulation of cells in the G0/G1 48.91% and 49.58% and from 24.02% to 35% and 37.69%, phase induced by DER in the presence of DOX is unknown. respectively. Also, our results show that DOX alone caused We suggest that the increase in the percentage of CMT-U27 late apoptosis rather than early apoptosis. However, the early cells in the G0/G1 phase may be related to the accompanying apoptotic cell number was significantly increased by the ad- changes in the level of proteins regulating the cell cycle and dition of DER (250 µM). We suggest that the increase in the the decline of cell proliferation activity. percentage of early apoptotic cells could be related to DER. In conclusion, we elucidated that DER enhanced the anti- DER may cause cell membrane loss and therefore allow proliferative effect of DOX in conjunction with induction of DOX to more easily penetrate into cells, and it may sensitize apoptosis by modulation of Bcl-2 expression and changes in mammary cancer cells to DOX by activating the apoptotic the cell cycle of the CMT-U27 cell line. Although the exact program. Also, the increases in apoptotic activity according molecular mechanism of the alterations in the cell cycle and to DOX concentration support this suggestion. In support apoptosis observed for DER and DOX combinations require of these possibilities, some reports have [44, 65] suggested further investigations, the results suggest that the synergistic that NSAIDs, as disordering agents on membranes, might effect of DOX and DER combinations in CMT therapy may interfere with the lateral heterogeneity of membranes, be achieved at relatively lower doses of DOX with lesser disrupting the organization and function of microdomains, side effects. which are involved in the regulation of protein location and signaling pathways. Alteration of membrane properties, such ACKNOWLEDGMENTS. This work was supported by as perturbations of membrane fluidity by anti-inflammatory grant no. 108O296 from the Scientific and Technological agents, has been recently described and has been indicated as Research Council of Turkey (TUBITAK) and by a grant from an additional mechanism by which anti-inflammatory agents the Scientific Research Projects Coordination Unit of Istan- have pharmacological effects relating to anticancer activity bul University (Project no. UDP-6853). The authors would in some publications [32, 65]. In this regard, previous studies like to thank Professor Eva Hellmén (Division of Anatomy have demonstrated that COX-2 inhibitors (e.g., celecoxib, and Physiology, Swedish University of Agricultural Scienc- indomethacin, nimesulide) augmented chemotherapeutic es, Sweden) for her kind donation of the CMT-U27 cell line. drug-induced apoptosis by downregulation of anti-apoptotic mediators, such as Bcl-2, in breast, prostate and osteosar- coma cells [13, 38, 69]. To define the mechanistic role of DER in apoptosis induction, we examined Bcl-2 expression, 666 T. BAKIREL ET AL.
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